S. aureus polypeptide and antibodies

ABSTRACT

The invention relates to antigenic polypeptides expressed by pathogenic microbes, vaccines comprising said polypeptides; therapeutic antibodies directed to said polypeptides and methods to manufacture said polypeptides, vaccines and antibodies.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/420,497, filed Mar. 14, 2012, now U.S. Pat. No. 8,632,779, issuedJan. 21, 2014, which is a divisional of U.S. application Ser. No.12/826,160, filed Jun. 29, 2010, now U.S. Pat. No. 8,163,288, issuedApr. 24, 2012, which is a continuation of U.S. application Ser. No.11/909,258, filed Jun. 12, 2008, now U.S. Pat. No. 7,767,211 issued Aug.3, 2010, which claims priority to International Application No.PCT/GB2006/000826, filed Mar. 8, 2006, which claims priority to UnitedKingdom Application No. GB0505949.8, filed Mar. 23, 2005, thedisclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to antigenic polypeptides expressed by pathogenicmicrobes, vaccines and immunogenic compositions comprising the antigenicpolypeptides and therapeutic antibodies directed to the antigenicpolypeptides.

BACKGROUND

A problem facing current medical development is the evolution ofantibiotic resistant strains of a number of significant pathogenicmicrobes. An example of a pathogenic organism which has developedresistance to antibiotics is Staphylococcus aureus. S. aureus is abacterium whose normal habitat is the epithelial lining of the nose inabout 20-40% of normal healthy people and is also commonly found onpeople's skin usually without causing harm. However, in certaincircumstances, particularly when skin is damaged, this germ can causeinfection. This is a particular problem in hospitals where patients mayhave surgical procedures and/or be taking immunosuppressive drugs. Thesepatients are much more vulnerable to infection with S. aureus because ofthe treatment they have received. Resistant strains of S. aureus havearisen in recent years. Methicillin resistant strains are prevalent andmany of these resistant strains are also resistant to several otherantibiotics. Currently there is no effective vaccination procedure forS. aureus.

The present invention is concerned with the identification of potentialvaccine components and therapies against which the problem of directlyresistant pathogen strains is avoided or reduced.

Amongst the approximately 4100 genes in the soil gram-positive bacteriumBacillus subtilis chromosome, 271 are indispensable (“essential”) forgrowth and among them, 23 have undefined roles in the physiology of theorganism (gcp, obg, ppaC-yybQ-, trmU, yacA, yacM, ydiB, ydiC, yjbN,ykqC, ylaN, yloQ, ylqF, ymdA, yneS, yphC, yqeH, yqeI, yqjK, yrvO, ysxC,ytaG, ywlC) (Kunst et al. 1997). Homologs of the proteins encoded bythese genes can be found in the various strains sequenced thus far ofanother gram-positive bacterium, the human pathogen Staphylococcusaureus. Amongst them, the Gcp and YneS orthologs are predicted membraneproteins, while the rest are predicted cytoplasmic proteins.Nonetheless, Obg has been shown to be partially bound to membranes in B.subtilis (Kobayashi et al. 2001).

SUMMARY

The inventors have isolated certain polypeptides that are essentialcomponents for growth of the pathogens Bacillus subtilis andStaphylococcus aureus and have raised antisera against thesepolypeptides. Antisera raised against the Bacillus subtilis polypeptideswas found to result in extremely potent killing of Staphylococcusaureus. This effect could not have been predicted. The present findingsfacilitate the development of vaccines, immunogenic compositions andantibody therapies that mitigate some of the problems of currenttherapies such as antibiotic resistance.

The present disclosure provides antigenic polypeptides that areessential for growth of the gram-positive bacteria Bacillus subtilis andStaphylococcus aureus and which are useful in the treatment orprevention of microbial infections.

According to a first aspect, there is provided an antigenic polypeptide,or part thereof, encoded by an isolated nucleic acid sequence selectedfrom the group consisting of:

-   -   i) a nucleic acid sequence as shown in FIGS. 1 to 6 (SEQ ID NO:        1-7);    -   ii) a nucleic acid sequence as in (i) which encodes a        polypeptide expressed by a pathogenic organism;    -   iii) a nucleic acid sequence which hybridizes to a sequence        identified in (i) or (ii) above; and    -   iv) a nucleic acid sequence that is degenerate as a result of        the genetic code to the nucleic acid sequence defined in        (i), (ii) or (iii)        for use as a medicament.

In one aspect, the medicament is a vaccine or immunogenic composition.

The nucleic acid encoding an antigenic polypeptide of the first aspectof the disclosure may anneal under stringent hybridization conditions toa nucleic acid sequence shown in FIGS. 1 to 6 (SEQ ID NO: 1-7) or to itscomplementary strand. Stringent hybridization/washing conditions arewell known in the art. For example, nucleic acid hybrids that are stableafter washing in 0.1×SSC, 0.1% SDS at 60° C. It is well known in the artthat optimal hybridization conditions can be calculated if the sequenceof the nucleic acid is known. For example, hybridization conditions canbe determined by the GC content of the nucleic acid subject tohybridization. Please see Sambrook et at (1989) Molecular Cloning; ALaboratory Approach. A common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified homology is:T _(m)=81.5° C.+16.6 Log [Na⁺]+0.41[% G+C]−0.63(% formamide).

The nucleic acid encoding the antigenic polypeptide of the first aspectof the invention may comprise a sequence set out in FIGS. 1 to 6 (SEQ IDNO: 1-7) or a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, for example 98%, or 99%, identical to a nucleic acid sequenceset out in FIGS. 1 to 6 (SEQ ID NO: 1-7) at the nucleic acid residuelevel.

“Identity”, as known in the art, is the relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, identity also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. Identity can be readily calculated(Computational Molecular Biology, Lesk, A. M. ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., AND Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or two polypeptide sequences, the term is well-known toskilled artisans (Sequence Analysis in Molecular Biology, von Heinje,G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonlyemployed to determine identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988). Preferred methods to determine identityare designed to give the largest match between the sequences tested.Methods to determine identity are codified in computer programs.Preferred computer program methods to determine identity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)),BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403 (1990)).

The nucleic acid encoding an antigenic polypeptide disclosed herein maycomprise a fragment of a sequence according which is at least 30 baseslong, for example, 40, 50, 60, 70, 80 or 90 bases in length.

The nucleic acid sequence encoding the antigenic polypeptide of thefirst aspect of the invention may be genomic DNA, cDNA or RNA, forexample mRNA.

The antigenic polypeptide of the first aspect of the invention may be acell membrane protein, for example an integral membrane protein or acytoplasmic protein.

Preferably, the antigenic polypeptide of the first aspect of theinvention is expressed by a pathogenic organism, for example, abacterium, virus or yeast. Preferably the pathogenic organism is abacterium. The bacterium may be a gram-positive or gram-negativebacterium, preferably a gram-positive bacterium. The bacterium may beselected from the group consisting of: Bacillus subtillis,Staphylococcus aureus; Staphylococcus epidermidis; Enterococcusfaecalis; Mycobacterium tuberculsis; Streptococcus group B;Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhea;Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis;Histoplasma sapsulatum; Neisseria meningitidis type B; Shigellaflexneri; Escherichia coli; Haemophilus influenzae; Listeriamonocytogenes, Bacillus anthracis, Corynebacterium diptheriae,Clostridium tetani, Mycoplasma spp. and Treponema pallidum. Preferablythe bacterium is of the genus Staphylococcus spp. Preferably still thebacterium is Staphylococcus aureus.

In a preferred embodiment of the invention, the antigenic polypeptide isassociated with infective pathogenicity of an organism as definedherein.

In a further preferred aspect of the invention the antigenic polypeptidecomprises all, or part of, an amino acid sequence shown in FIGS. 7 to 12(SEQ ID NO: 8-14). As used herein “part of” may include a polypeptidefragment which may be at least 10, 15, 20 or 30 amino acids long. Theantigenic polypeptide may comprise a non-protein antigen, for example apolysaccharide antigen.

As used herein, the term “polypeptide” means, in general terms, aplurality of amino acid residues joined together by peptide bonds. It isused interchangeably and means the same as peptide, protein,oligopeptide, or oligomer. The term “polypeptide” is also intended toinclude fragments, analogues and derivatives of a polypeptide whereinthe fragment, analogue or derivative retains essentially the samebiological activity or function as a reference protein.

According to a second aspect of the invention there is provided a vectorcomprising a nucleic acid sequence encoding a polypeptide disclosedherein.

The vector of the second aspect of the invention may be a plasmid,cosmid or phage. The vector may include a transcription control sequence(promoter sequence) which mediates cell specific expression, forexample, a cell specific, inducible or constitutive promoter sequence.The vector may be an expression vector adapted for prokaryotic oreukaryotic gene expression, for example, the vector may include one ormore selectable markers and/or autonomous replication sequences whichfacilitate the maintenance of the vector in either a eukaryotic cell orprokaryotic host (Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. andreferences therein; Marston, F (1987) DNA Cloning Techniques: APractical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F MAusubel et al, Current Protocols in Molecular Biology, John Wiley &Sons, Inc. (1994). Vectors which are maintained autonomously arereferred to as episomal vectors.

Promoter is an art recognized term and may include enhancer elementswhich are cis acting nucleic acid sequences often found 5′ to thetranscription initiation site of a gene (enhancers can also be found 3′to a gene sequence or even located in intronic sequences and istherefore position independent). Enhancer activity is responsive totrans acting transcription factors (polypeptides) which have been shownto bind specifically to enhancer elements. The binding/activity oftranscription factors (see Eukaryotic Transcription Factors, by David SLatchman, Academic Press Ltd, San Diego) is responsive to a number ofenvironmental cues which include intermediary metabolites (eg glucose,lipids), environmental effectors (e.g. light, heat).

Promoter elements also include so called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation. These sequences also bind polypeptides whichfunction, inter alia, to facilitate transcription initiation selectionby RNA polymerase.

The vector of the second aspect of the invention may include atranscription termination or polyadenylation sequences. This may alsoinclude an internal ribosome entry sites (IRES). The vector may includea nucleic acid sequence that is arranged in a bicistronic ormulti-cistronic expression cassette.

According to a third aspect of the invention there is provided a methodfor the production of a recombinant antigenic polypeptide disclosedherein comprising:

-   -   (i) providing a cell transformed/transfected with a vector        according to the second aspect of the invention;    -   (ii) growing said cell in conditions suitable for the production        of said polypeptides; and    -   (iii) purifying said polypeptide from said cell, or its growth        environment.

In a preferred aspect of the method of the third aspect, the vectorencodes, and thus said recombinant polypeptide is provided with, asecretion signal to facilitate purification of said polypeptide.

According to a fourth aspect of the invention there is provided a cellor cell-line transformed or transfected with the vector according to thesecond aspect of the invention. In a preferred embodiment, said cell isa prokaryotic cell, for example, yeast or a bacterium such as E. coli.

Alternatively said cell is a eukaryotic cell, for example a fungal,insect, amphibian, mammalian, for example, COS, CHO cells, BowesMelanoma and other suitable human cells, or plant cell.

According to a fifth aspect of the invention there is provided a vaccineor immunogenic composition comprising at least one antigenicpolypeptide, or part thereof, according to the first aspect of theinvention. Preferably said vaccine or immunogenic composition furthercomprises a carrier and/or adjuvant. As used herein “part thereof” mayinclude a fragment or subunit of the antigenic polypeptide wherein thefragment or subunit is sufficient to induce an antigenic response in arecipient.

The vaccine or immunogenic composition according to the fifth aspect maybe a subunit vaccine or immunogenic composition in which the immunogenicpart of the vaccine or immunogenic composition is a fragment or subunitof the antigenic polypeptide according to the first aspect of theinvention.

The terms adjuvant and carrier are construed in the following manner.Some polypeptide or peptide antigens contain B-cell epitopes but no Tcell epitopes. Immune responses can be greatly enhanced by the inclusionof a T cell epitope in the polypeptide/peptide or by the conjugation ofthe polypeptide/peptide to an immunogenic carrier protein such as keyhole limpet haemocyanin or tetanus toxoid which contain multiple T cellepitopes. The conjugate is taken up by antigen presenting cells,processed and presented by human leukocyte antigens (HLA's) class IImolecules. This allows T cell help to be given by T cell's specific forcarrier derived epitopes to the B cell which is specific for theoriginal antigenic polypeptide/peptide. This can lead to increase inantibody production, secretion and isotype switching.

An adjuvant is a substance or procedure which augments specific immuneresponses to antigens by modulating the activity of immune cells.Examples of adjuvants include, by example only, agonistic antibodies toco-stimulatory molecules, Freunds adjuvant, muramyl dipeptides, andliposomes. An adjuvant is therefore an immunomodulator. A carrier is animmunogenic molecule which, when bound to a second molecule augmentsimmune responses to the latter.

In yet a further aspect of the invention there is provided a method toimmunize an animal against a pathogenic microbe comprising administeringto said animal at least one polypeptide, or part thereof, according tothe first aspect of the invention. Preferably, the polypeptide is in theform of a vaccine or immunogenic composition according to the fifthaspect of the invention. In a preferred method of the invention theanimal is human.

Preferably the antigenic polypeptide of the first aspect, or the vaccineor immunogenic composition of the fifth aspect, of the invention can bedelivered by direct injection either intravenously, intramuscularly,subcutaneously. Further still, the vaccine or antigenic polypeptide, maybe taken orally. The polypeptide or vaccine may be administered in apharmaceutically acceptable carrier, such as the various aqueous andlipid media, such as sterile saline, utilized for preparing injectablesto be administered intramuscularly and subcutaneously. Conventionalsuspending and dispersing agents can be employed. Other means ofadministration, such as implants, for example a sustained low dosereleasing bio-observable pellet, will be apparent to the skilledartisan.

The vaccine may be against the bacterial species Staphylococcus aureusS. epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, B.anthracis, and/or Listeria monocytogenes.

It will also be apparent that vaccines or antigenic polypeptides areeffective at preventing or alleviating conditions in animals other thanhumans, for example and not by way of limitation, family pets (e.g.domestic animals such as cats and dogs), livestock (e.g. cattle, sheep,pigs) and horses.

A further aspect of the invention provides a pharmaceutical compositioncomprising an effective amount of at least one of the polypeptides ofthe invention, or a vaccine or immunogenic composition of the invention.These polypeptides may also include a pharmaceutically acceptablecarrier or diluent.

According to a further aspect of the invention there is provided anantibody, or at least an effective binding part thereof, which binds atleast one antigenic polypeptide, or part thereof, according to theinvention.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any binding member or substance having abinding domain with the required specificity for the antigenicpolypeptide. Thus, this term covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including anypolypeptide comprising an immunoglobulin binding domain, whether naturalor wholly or partially synthetic. Chimeric molecules comprising animmunoglobulin binding domain, or equivalent, fused to anotherpolypeptide are therefore included. Cloning and expression of chimericantibodies are described in EP-A-0120694 and EP-A-0125023.

In a preferred aspect of the invention said antibody is a polyclonal ormonoclonal antibody.

In a further preferred aspect of the invention said antibody is achimeric antibody produced by recombinant methods to contain thevariable region of said antibody with an invariant or constant region ofa human antibody.

In a further preferred aspect of the invention, said antibody ishumanized by recombinant methods to combine the complementaritydetermining regions of said antibody with both the constant (C) regionsand the framework regions from the variable (V) regions of a humanantibody.

Preferably said antibody is provided with a marker including aconventional label or tag, for example a radioactive and/or fluorescentand/or epitope label or tag.

Preferably said humanized monoclonal antibody to said polypeptide isproduced as a fusion polypeptide in an expression vector suitablyadapted for transfection or transformation of prokaryotic or eukaryoticcells.

Antibodies, also known as immunoglobulins, are protein molecules whichhave specificity for foreign molecules (antigens). Immunoglobulins (Ig)are a class of structurally related proteins consisting of two pairs ofpolypeptide chains, one pair of light (L) (low molecular weight) chain(κ or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all fourlinked together by disulphide bonds. Both H and L chains have regionsthat contribute to the binding of antigen and that are highly variablefrom one Ig molecule to another. In addition, H and L chains containregions that are non-variable or constant.

The L chains consist of two domains. The carboxy-terminal domain isessentially identical among L chains of a given type and is referred toas the “constant” (C) region. The amino terminal domain varies from Lchain to L chain and contributes to the binding site of the antibody.Because of its variability, it is referred to as the “variable” (V)region.

The H chains of Ig molecules are of several classes, α, μ, σ, α, and γ(of which there are several sub-classes). An assembled Ig moleculeconsisting of one or more units of two identical H and L chains derivesits name from the H chain that it possesses. Thus, there are five Igisotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on thedifferences in the H chains, i.e., IgG1, IgG2, IgG3 and IgG4). Furtherdetail regarding antibody structure and their various functions can befound in, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press.

Chimeric antibodies are recombinant antibodies in which all of theV-regions of a mouse or rat antibody are combined with human antibodyC-regions. Humanized antibodies are recombinant hybrid antibodies whichfuse the complementarity determining regions from a rodent antibodyV-region with the framework regions from the human antibody V-regions.The C-regions from the human antibody are also used. The complementaritydetermining regions (CDRs) are the regions within the N-terminal domainof both the heavy and light chain of the antibody to where the majorityof the variation of the V-region is restricted. These regions form loopsat the surface of the antibody molecule. These loops provide the bindingsurface between the antibody and antigen.

Antibodies from non-human animals provoke an immune response to theforeign antibody and its removal from the circulation. Both chimeric andhumanized antibodies have reduced antigenicity when injected to a humansubject because there is a reduced amount of rodent (i.e. foreign)antibody within the recombinant hybrid antibody, while the humanantibody regions do not illicit an immune response. This results in aweaker immune response and a decrease in the clearance of the antibody.This is clearly desirable when using therapeutic antibodies in thetreatment of human diseases. Humanized antibodies are designed to haveless “foreign” antibody regions and are therefore thought to be lessimmunogenic than chimeric antibodies.

In a further preferred embodiment of the invention said antibodies areantibodies whose activity is mediated by complement, for example theactivity of the antibody may be activated by complement.

In another aspect of the invention there is provided a vector comprisinga nucleic acid sequence encoding the humanized or chimeric antibodiesaccording to the invention.

In a yet further aspect of the invention, there is provided a cell orcell line which comprises the vector encoding the humanized or chimericantibody according to the invention. The cell or cell line may betransformed or transfected with the vector encoding the humanized orchimeric antibody according to the invention.

In a yet further aspect of the invention there is provided a hybridomacell line which produces a monoclonal antibody as hereinbeforedescribed.

In a further aspect of the invention there is provided a method ofproducing monoclonal antibodies according to the invention usinghybridoma cell lines according to the invention.

In a yet further aspect of the invention there is provided a method forthe production of the humanized or chimeric antibody according to theinvention comprising:

-   -   (i) providing a cell transformed or transfected with a vector        which comprises a nucleic acid molecule encoding the humanized        or chimeric antibody according to the invention;    -   (ii) growing said cell in conditions suitable for the production        of said antibody; and    -   (iii) purifying said antibody from said cell, or its growth        environment.

In a further aspect of the invention there is provided a method forpreparing a hybridoma cell-line according to the invention comprisingthe steps of:

-   -   i) immunizing an immunocompetent mammal with an immunogen        comprising at least one polypeptide having an amino acid        sequence as represented in FIGS. 7 to 12 (SEQ ID NO: 8-14), or        fragments thereof;    -   ii) fusing lymphocytes of the immunized immunocompetent mammal        with myeloma cells to form hybridoma cells;    -   iii) screening monoclonal antibodies produced by the hybridoma        cells of step (ii) for binding activity to the amino acid        sequences of (i);    -   iv) culturing the hybridoma cells to proliferate and/or to        secrete said monoclonal antibody; and    -   v) recovering the monoclonal antibody from the culture        supernatant.

The immunocompetent mammal may be a mouse, rat or rabbit.

The production of monoclonal antibodies using hybridoma cells iswell-known in the art. The methods used to produce monoclonal antibodiesare disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) andalso by Donillard and Hoffman, “Basic Facts about Hybridomas” inCompendium of Immunology V.II ed. by Schwartz, 1981, which areincorporated by reference.

In a further aspect of the invention there is provided the use of anantigenic polypeptide according to the first aspect of the invention inthe manufacture of a medicament for the treatment or prophylaxis of amicrobial infection or a microbe related disorder.

Preferably, the microbial infection is a bacterial infection caused by abacterial pathogen derived from a bacterial species selected from thegroup consisting of: Staphylococcus spp e.g. Staphylococcus aureus,Staphylococcus pyrogenes, Staphylococcus epidermidis; Enterococcus sppe.g. Enterococcus faecalis; Lysteria spp; Pseudomonas spp; Mycobacteriumspp e.g. Mycobacterium tuberculsis; Enterobacter spp; Campylobacter spp;Salmonella spp; Streptococcus spp, e.g. Streptococcus group A or B,Streptoccocus pneumoniae; Helicobacter spp, e.g. Helicobacter pylori;Neisseria spp e.g. Neisseria gonorrhea, Neisseria meningitidis; Borreliaburgdorferi spp; Shigella spp, e.g. Shigella flexneri; Escherichia colispp; Haemophilus spp, e.g. Haemophilus influenza; Chlamydia spp e.g.Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci;Francisella tularensis; Bacillus spp, e.g. Bacillus anthracis;Clostridia spp, e.g. Clostridium botulinum; Yersinia spp, e.g. Yersiniapestis; Treponema spp; and Burkholderia spp, e.g. Burkholderia malleiand B. pseudomallei.

The bacteria related disorder may be a Staphylococcus aureus-associateddisorder. A Staphylococcus aureus-associated disorder may include, forexample, septicaemia; tuberculosis; bacteria-associated food poisoning;blood infections; peritonitis; endocarditis; osteomyelitis; sepsis; skindisorders, meningitis; pneumonia; stomach ulcers; gonorrhoea; strepthroat; streptococcal-associated toxic shock; necrotizing fasciitis;impetigo; histoplasmosis; Lyme disease; gastro-enteritis; dysentery; andshigellosis

In a further aspect of the invention there is provided the use ofantibodies according to the invention in the manufacture of a medicamentfor the treatment of a microbial infection.

In a further aspect of the invention there is provided a method oftreating a patient comprising administering to the patient an antigenicpolypeptide according to the first aspect of the invention, or a vaccineor immunogenic composition according to the fifth aspect of theinvention, or an antibody according to the invention.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

An embodiment of the invention will now be described by example only andwith reference to the following materials, methods and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence of the yphC polypeptide from Bacillussubtilis (SEQ ID NO: 1);

FIG. 2 shows the DNA sequence of the ysxC polypeptide from Bacillussubtilis (SEQ ID NO: 2);

FIG. 3 shows the DNA sequence of the ywlC polypeptide from Bacillussubtilis (SEQ ID NO: 3);

FIG. 4 shows the DNA sequence of the yneS ortholog peptide 731 fromStaphylococcus aureus (SEQ ID NO: 4);

FIG. 5 shows the DNA sequence of the yneS ortholog peptide 733 fromStaphylococcus aureus (SEQ ID NO: 5);

FIG. 6 shows (a) the DNA sequence encoding the gcp region putativelyexposed outside of the membrane (SEQ ID NO: 6); and (b) the full DNAsequence of the gcp ortholog polypeptide, both from Staphylococcusaureus (SEQ ID NO: 7);

FIGS. 7 to 11 show the amino acid sequences (SEQ ID NO: 8-12)corresponding to the DNA sequences shown in FIGS. 1 to 5 (SEQ ID NO:1-5) respectively;

FIGS. 12( a) and (b) show the amino acid sequences (SEQ ID NO: 13 and14) corresponding to the DNA sequences shown in FIGS. 6 (a) and (b) (SEQID NO: 6 and 7) respectively;

FIGS. 13 and 14 show hydropathy plots of the membrane proteins yneS andgcp. The calculation of the hydropathy plots of the proteins statedabove and the corresponding graphic representation to predict thetransmembrane topology model was determined according to the ConPredIIMethod and was carried in the serverhttp://bioinfo.si.hirosaki-u.ac.jp/˜ConPred2/;

FIG. 15 shows graphs showing that heat treatment of sera from a humanpatient (□), from a non-immunized rabbit (◯) or from sera raised againstthe A. thaliana cyclophilin protein (Δ) did not induce death of S.aureus SJF741. No killing of S. aureus SJF741 was observed either whenusing native sera from a patient convalescent from S. aureus infection(▪) (Panel A) and from a non-immunized rabbit (●) (Panel B). When nativesera raised against the A. thaliana cyclophilin protein (▴) (Panel C),against the B. subtilis proteins Obg (▾) and YdiB (

) (Panel D) and against the S. aureus protein SA1387 (♦) (Panel E) aminor decrease in the number of S. aureus SJF741 during the first 6hours was observed, which was followed by subsequent recovery.

FIG. 16 shows graphs showing that native sera raised against the B.subtilis proteins YsxC (●), YphC (▪), and YwlC (▴) (Panels A and B)killed S. aureus SJF471 dramatically, a 5 log decrease within 2 to 4hours. A similar effect was observed when using native sera raisedagainst the S. aureus peptides YneS-731 (▾) and YneS 733 (♦) and the S.aureus protein Gcp (

) (Panels C-E). In contrast, heat treating the sera raised against theB. subtilis YsxC protein (◯) or the S. aureus peptides YneS-731 (∇) andYneS-733 (⋄) (Panels A, C, D) abolished the killing abilities of thesesera, which were able to kill S. aureus SJF741 in the native form (notheat-treated), as indicated above. Hence, the killing abilities of thesera are due to a heat labile component, which is inactivated in theheat treated sample. No experiments using heat treated sera raisedagainst the B. subtilis proteins YphC (▪) and YwlC (▴) or against the S.aureus gcp protein (

) are shown in this figure, and the experiments with the correspondingnative sera (Panels B and E), as indicated above, illustrate the S.aureus killing capability of these sera.

DETAILED DESCRIPTION

Materials and Methods

Strains

The chromosomal DNA used for PCR amplification of the gene sequences ofinterest were B. subtilis subsp. subtilis str. 168, S. aureus NCTC 8325,S. aureus N315 and S. aureus COL. An erythromycin resistant sodA::lacZtranscriptional fusion derivative of S. aureus SH1000 (S. aureusSJF741), was the strain used in the assays (Horsburgh et al. 2002).

DNA, Protein and Peptide Sequences Used as Antigens.

The gene and protein sequences of the genes mentioned can be found at:B. subtilis subsp. subtilis str. 168: GenBank Accession AL009126;

S. aureus 8325 (this is a non-annotated sequence; equivalent annotatedsequences of S. aureus containing the genes of interest can be foundbelow): Iandolo et al., 2002; Novick, 1967;

Other S. aureus Strains:

S. aureus subsp aureus str. N315: Kuroda, 2001;

S. aureus strain subsp. aureus COL: The Center for Genomic Research;NCBI Taxonomy Database, Taxonomy ID 93062

NOTE: Different strains of S. aureus have different locus names for thesame genes due to phage insertions within the sequence. In thisdocument, the locus names used for the S. aureus genes correspond tothose in the S. aureus N315 sequence.

Antigen Preparation

The genes encoding selected proteins from Bacillus subtilis 168 (Obg,YdiB, YphC (FIG. 1; SEQ ID NO: 1), YsxC (FIG. 2; SEQ ID NO: 2), YwlC(FIG. 3; SEQ ID NO: 3), and S. aureus N315 (SA1387, Gcp/SA1854 (FIG. 6;SEQ ID NO: 6 and 7)) were amplified by PCR. The resulting products werecloned in plasmid pETBlue-1, and the genes overexpressed in Escherichiacoli Tuner™ (DE3) pLacI Competent Cells (Novagen) according to themanufacturer's instructions. The overexpressed proteins were purified ina 3-step scheme based on anion exchange, hydrophobic and gel filtrationchromatography. The level of protein overexpression was confirmed bySDS-PAGE, and the purity had an average of 90%. In addition, selectedpeptides within the S. aureus N315 protein SA1187 (YneS-731 (FIG. 4; SEQID NO: 4) and YneS-733 (FIG. 5; SEQ ID NO: 5)) were synthesized on aMilligen 9050 Peptide Synthesizer using F-moc chemistry. The F-moc aminoacids (Novobiochem/Merck) were activated immediately before couplingusing equimolar amounts of HCTU or HBTU in the presence of a 10% molarexcess of HOBt. In both cases, a cysteine was incorporated at theC-terminus of the peptide to enable linkage to carrier protein byassembling the peptide on Fmoc-L-Cys(Trt)-PEG-PS resin (AppliedBiosystems). Peptides were purified using a C18 Vydac column (22×250 mm)using gradients of acetonitrile in 0.1% TFA. Peptides were verified bymass spectrometry. The purified peptides were conjugated to KLH (Sigma)(carrier protein) to enhance immunogenicity of the hapten in the rabbit.Conjugation was performed in 10× PBS using MBS (Sigma).

Sera

Sera were obtained from the Antibody Resource Center at the Universityof Sheffield from: i) rabbits immunized against proteins from B.subtilis (Obg, YdiB, YphC, YwlC and YsxC and S. aureus (Gcp, SA1387);ii) rabbits immunized against KLH-conjugated peptides selected withinthe S. aureus protein SA1187 (YneS-731, YneS-733); iii) rabbitsimmunized against a KLH-conjugated peptide from the cyclophilin proteinfrom Arabidopsis thaliana; iv) naive (non-immune) rabbit serum; and v)human serum from a patient convalescent from a S. aureus infection.

The immunization process was performed as follows. For each rabbit 200to 500 μg of antigen (in a maximum volume of 250 ul of Phosphate BufferSaline, PBS) were mixed with an equal volume of complete Freund'sadjuvant. The solution was filtered through a 23G needle until anemulsion formed which did not separate on standing. Each rabbit wasinoculated with a maximum of 500 μl subcutaneously. On day 22, 43 and 64the injection was repeated but using incomplete Freund's adjuvant.Sample bleeds were collected on day 53 and after day 64. Injection dateswere flexible within a range of 3 to 6 weeks. When a suitable titer wasdetected in the test serum, a final boost followed by bleed out 10 dayslater was performed.

Sera were stored frozen being thawed and filtered through 0.2 μm porediameter filters (Minisart High Flow, Sartorius) immediately before usein killing experiments.

Using western blot analysis (data not shown) it was shown thatantibodies against the B. subtilis YdiB recognize a band of the sizecorresponding to the YdiB homolog in S. aureus, suggesting the speciescross-reactivity of these antibodies.

Media and Growth Conditions

To prepare the inoculum for the serum experiments, S. aureus SJF741 wasgrown at 37° C. in Brain Heart Infusion medium (BHI; Oxoid) supplementedwith erythromycin (Sigma) to a final concentration of 5 μg/ml (BHI-Ery).

Preparation of the Inoculum

A single colony of S. aureus SJF741 freshly grown on BHI-Ery plates fromthe laboratory frozen stock was inoculated in 30 ml universalscontaining 5 ml of BHI-Ery and incubated overnight (between 12 to 16hours) at 37° C. in an orbital shaker (250 rpm). A 10-fold dilution inPhosphate Saline Buffer (PBS) of the resulting culture was preparedimmediately before inoculation into serum.

Serum Experiments

Aliquots of 200 μl from the various sera in 1.5 ml microfuge tubes wereinoculated with the PBS dilution of S. aureus SJF741 (See Preparation ofthe inoculum) to a final cell density of 1×10⁶ to 1×10⁷ cells/ml,followed by incubation in a rotary shaker at 37° C. 10 ul samples weretaken periodically from these serum cultures, serially diluted, and 10ul from each dilution plated on BHI-Ery plates, which were subsequentlyincubated at 37° C. overnight. In addition, another 10 ul sample fromeach serum culture was directly plated on BHI-Ery plates. Only thedilutions rendering between 1 to 40 colonies were enumerated and thenumber of viable cells (colony forming units, CFU) per ml determined.

Results

To evaluate the staphylococcal killing abilities of the various sera, S.aureus was challenged with the various rabbit anti-sera and survivalover time was evaluated. The results showed that S. aureus wasdramatically killed within 2 to 3 hours of contact with sera (FIG. 16)containing antibodies against Gcp and YneS, as well as to other surfaceproteins. In contrast, antibodies against cytoplasmic proteins from B.subtilis (Obg and YdiB), to a membrane protein from Arabidopsis thaliana(cyclophilin), and to various normal rabbit sera did not show thebactericidal phenotype (FIG. 15). Strikingly, sera from rabbitsimmunized against other presumed cytoplasmic proteins from B. subtilis(YsxC and YphC and YwlC) also revealed a killing phenotype similar tothe one observed for Gcp and YneS (731 and 733) antibodies. This wasunexpected since YsxC, YphC and YwlC are presumed cytoplasmic proteinsand, therefore, are not surface exposed and so the antisera would not beexpected to recognize them.

This work suggests the location of YsxC in the membrane fraction of S.aureus. This work has further demonstrated that the killing effect ismediated through a heat-labile component (inactivated by heat treatment,See Material and Methods) present in serum, likely to correspond to someof the components of the complement (FIG. 16).

REFERENCES

-   Horsburgh et al., J. Bacteriol. 184(9):5457-67 (2002)-   Iandolo et al., Gene 289 109-118 (2002).-   Ikeda et al., In Silico Biol., 2, 19-33 (2002).-   Ikeda et al., Nucleic Acids Res., 31, 406-409 (2003).-   Karavolos et al., Microbiology October; 149(Pt 10):2749-58 (2003).-   Kobayashi et al., Mol Microbiol. September; 41(5):1037-51 (2001).-   Kobayashi et al. Proc Natl Acad Sci USA 100(8):4678-83 (2003).-   Kunst et al., Nature, November 20; 390(6657):249-56 (1997).-   Kuroda et al. Lancet, 357:1225-1240 (2001).-   Lao and Shimizu In Valafar, F. (ed.), Proceedings of the 2001    International Conference on Mathematics and Engineering Techniques    in Medicine and Biological Sciences (METMBS '01), CSREA Press, USA,    pp. 119-125 (2001).-   Lao et al., Bioinformatics, 18, 562-566 (2002).-   Lao et al., In Silico Biol., 2, 485-494 (2002).-   Moszer et al., Nucleic Acids Res. 30(1):62-5 (2002).-   Novick, R. P. Virology 33:155-156 (1967).-   Xia et al., Comput. Biol. Chem., 28, 51-60 (2004).-   Zalacain et al., J Mol Microbiol Biotechnol. 6(2):109-26 (2003).

We claim:
 1. An isolated monoclonal, humanized or chimeric antibody, oran effective binding part thereof, which binds an isolated antigenicpolypeptide encoded by an isolated nucleic acid consisting of SEQ ID NO:6.
 2. The antibody of claim 1, wherein chimeric antibody is produced byrecombinant methods to contain the variable region of said antibody withan invariant or constant region of a human antibody.
 3. The antibody ofclaim 1, wherein the humanized antibody is humanized by recombinantmethods to combine the complementarity determining regions of saidantibody with both the constant (C) regions and the framework regionsfrom the variable (V) regions of a human antibody.
 4. The antibodyaccording to claim 1 wherein the antibody is an opsonic antibody.
 5. Amethod for preparing a hybridoma cell-line comprising the steps of: i)immunizing an immunocompetent mammal with a polypeptide consisting of anamino acid sequence of SEQ ID NO: 13; ii) fusing lymphocytes of theimmunized immunocompetent mammal with myeloma cells to form hybridomacells; iii) screening monoclonal antibodies produced by the hybridomacells of step (ii) for binding activity to the polypeptide of i); iv)culturing the hybridoma cells to proliferate and/or to secrete saidmonoclonal antibody; and v) recovering the monoclonal antibody from theculture supernatant.
 6. An isolated monoclonal, humanized or chimericantibody, or an effective binding part thereof, which binds an isolatedantigenic polypeptide consisting of SEQ ID NO:
 13. 7. The antibody ofclaim 6, wherein the chimeric antibody is produced by recombinantmethods to contain the variable region of said antibody with aninvariant or constant region of a human antibody.
 8. The antibody ofclaim 6, wherein humanized antibody is humanized by recombinant methodsto combine the complementarity determining regions of said antibody withboth the constant (C) regions and the framework regions from thevariable (V) regions of a human antibody.
 9. The antibody according toclaim 6 wherein said antibody is an opsonic antibody.